1. Field of the Invention
The present invention relates to a high-yield production system for a protein or peptide, and more specifically it relates to a production system using a regulator sequence derived from Trichoderma viride, and to a production technique for a protein or peptide which employs the production system.
2. Description of the Related Art
Filamentous fungi are known to secrete significant amounts of extracellular proteins, particular enzymes. For example, the genus Aspergillus secretes amylases, proteases, lipases, cellulases and other enzymes, which are therefore utilized in various different fields. As to their yields, it has been reported, for example, that Aspergillus niger produces over 20 g of glucoamylase per liter of liquid culture, while Aspergillus oryzae produces about 50 g per kilogram of solid culture (Katuya Gomi: Kagaku to Seibutsu (1994), 32, 269).
Recent years have brought an accumulation of knowledge regarding production techniques for target proteins utilizing the protein-producing abilities of these filamentous fungi. Examples of filamentous fungi-derived foreign proteins which have been produced include Mucor miehei-derived rennin in Aspergillus nidulans hosts (G. L. Gray, et al.; Gene (1986), 48, 41), Aspergillus ficuum-derived phytase in Aspergillus niger hosts (R. F. M. van Gorcom, et al.: European Patent Application (1991), 0420358A1), Mucor miehei-derived rennin (T. Christensen, et al.: Bio/Technology (1988), 6, 1419) and lipase (B. Huge-Jensen, et al.: Lipids (1989), 24, 781) in Aspergillus oryzae hosts, Phlebia radiata-derived laccase in Trichoderma reesei hosts (M. Saloheimo, et al.: Bio/Technology (1991), 9, 987), Aspergillus oryzae-derived .alpha.-amylase in Trichoderma viride hosts (C. Cheng, et al.: Agric. Biol. Chem. (1991), 55, 1817) and Fusarium genus-derived alkali proteases in Acremonium chrysogenum hosts (Shigeru Morita, et al.: Summary of Meeting Lectures of the Nihon Nogei Kagakukai (1993), p.140). Production of human, bovine and other animal proteins as well as plant proteins has also been confirmed in Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae and Trichoderma reesei hosts.
Thus, filamentous fungi are clearly excellent as production hosts for proteins and polypeptides. The productivity of the target protein also becomes an important issue in terms of industrial applications. The factors considered to be important in determining productivity include (1) regulation for effective and high-yield transcription and translation of the target protein by a regulator region (for example, promoter and terminator) which is capable of expression in the host, (2) a translation product with the desired higher-order structure (activity type) and (3) stable extracellular secretion of the same. To this end there have been developed many effective promoters, such as the Aspergillus amylase gene promoter and the Trichoderma cellulase gene promoter. It is currently known that the use of an .alpha.-amylase gene promoter in an Aspergillus oryzae host can produce 3.3 g of Mucor miehei rennin per liter of liquid culture.
No matter how powerful the promoter, however, it is usually not possible to obtain the target protein in an amount exceeding the protein yield of the host. In fact, only 30% protein productivity is achieved in the case of Aspergillus oryzae mentioned above. This is thought to be due to the fact that the gene which is used remains in the host, that the codon use frequency in the translated region differs among species, that the secretion mechanism differs among species, and other reasons as well, but as yet no technique has been discovered to solve these problems.
It has therefore been considered that target protein yields could be enhanced by improving the protein production of the hosts themselves.
Filamentous fungi belonging to the genus Trichoderma are known to be excellent cellulase producing cells. In particular, Trichoderma reesei has been widely studied in terms of its foreign protein production, and much research is being conducted on foreign protein expression using a promoter for the cellobiohydrolase 1 (cbh1) gene, said to constitute about 70% of the secreted protein of that species (Uusitalo J M., et al.: J. Biotechnol. (1991), 17, 35. Joutsjoki V V., et al.: Curr. Genet. (1993), 24, 223. Barnett C C., et al.: Biotechnology (1991), 9, 562. Berges T., et al.: Curr. Genet. (1993), 24, 53. Saloheimo M., et al.: Gene (1989), 85, 343. Saarelainen R., et al.: Mol. Gen. Genet. (1993), 241, 497). However, only C. Cheng et al. have reported a foreign protein expression system using Trichoderma viride as the host. According to C. Cheng et al., the .alpha.-amylase gene was introduced into a protease-deficient strain of Trichoderma viride as the host using the cbh1 promoter and signal sequence, to produce .alpha.-amylase at 1 g per liter of liquid culture. While this demonstrates that foreign protein production is possible with Trichoderma viride, that production level is not satisfactory in terms of cost on a practical production scale.